CN114796435B - 具有抗SARS-CoV-2病毒作用的口鼻用药物组合物及其制备方法 - Google Patents
具有抗SARS-CoV-2病毒作用的口鼻用药物组合物及其制备方法 Download PDFInfo
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Abstract
本发明涉及医药领域，具体而言，涉及具有防治SARS‑CoV‑2作用的口鼻用药物组合物及其制备方法。所述的药物组合物以重量份计，活性成分包括如下药物挥发油：广藿香油15～30份、丁香油10～20份、草果油1～5份、生姜油10～20份以及益智仁油10～20份。
Description
技术领域
本发明涉及医药领域，具体而言，涉及具有防治SARS-CoV-2作用的口鼻用药物组合物及其制备方法。
背景技术
据《中国疫病史鉴》记载，在传染病肆虐期间，芳香类中药由于具有辟秽解毒的作用，因此是驱除疫病最常用的一类中药。汉武帝时期发生瘟疫流行，人们靠熏烧芳香类中药抑制瘟疫的传播。唐宋时期的《备急千金要方》中记载了大量的芳香类中药和组方，当中包括辟瘟气的重要方剂，如太乙流金散和雄黄丸等；同时还记载了用艾烟熏蒸的方法可防治时气瘴疫。至明代的《本草纲目》更将芳香类中药分为芳草类(共56种，如荆芥和薄荷等)和香木类(共35种，如乳香和檀香等)；并提及于房间熏烧苍术和艾叶等来进行“空气消毒”。自从2003年以来，中医药在抗击SARS、MERS、EBOV等病毒性疾病过程中，已发挥特色优势作用，做出举世公认的重要贡献。国家中医药管理局于2009年发布的《甲型H1N1流感中医药预防方案》建议把芳香化浊中药，如苍术、艾叶、藿香、当归、白芷等，制成香囊或香熏，具有除瘴避秽的作用。国家卫生健康委员会于2020年发布的《新型冠状病毒感染的肺炎诊疗方案(试行第七版)》有关中医治疗方案中，医学观察期、确诊患者的临床治疗期及恢复期均选用含较多芳香类中药的中成药或方剂进行防治，同时部分地方卫生健康委员会或中医药管理局还推荐多种其他使用芳香类中药的方式(外熏、艾灸、佩戴香囊等)来防治新冠病毒。
目前尚无有效的抗新型冠状病毒药物，部分化学抗病毒药物虽有一定的抗病毒作用，但其局限性也很突出，如副作用和耐药性。而中药在预防病毒、细菌等引起的感染性疾病方面有独特的优势，药用资源丰富，临床应用历史悠久，辨证论治精准治疗，可多靶点作用于疾病，耐药性小，古今疫方常用芳香、辛散、清热药以散邪逐湿除热，特别在瘟疫(各种烈性传染病)的防治方面积累了大量珍贵的经验。近年来，诸多研究工作挖掘了中药对呼吸系统传染性疾病治疗的确定性和广泛性。现代医学证明中药里的挥发油可以抑制病毒蛋白转译或干扰病毒膜融合过程，从而阻止流感病毒进入宿主细胞，具有抗菌、抗病毒作用。此外，研究更表明挥发油能通过气态形式，并于短暂的暴露时间内(5-30分钟)起到杀菌弒病毒的功效。中药挥发油在预防和治疗病毒性疾病方面有着悠久的历史和独特的优势，尤其面对目前肆虐的新型冠状病毒更显示出良好的疗效和潜力，而且安全性高、适用范围广，是预防和治疗新型冠状病毒感染的重要资源。
鼻为肺之门户，而且病毒多通过口鼻而入，感染呼吸道及肺部。把具有抗新型冠状病毒作用的单方或复方中药挥发油制备成鼻腔保护剂或漱口水等个人防护产品，可利用挥发油自动挥发在鼻腔、咽喉和气管内形成高浓度气态小环境而快速弑灭病毒，同时通过鼻腔粘膜的药物层杀灭沉积在鼻腔的病毒并抑制其对细胞的入侵，从而形成口鼻的有效防线。尤其适用于封闭场所，达到全方位、全时段的保护。
公开号为CN111184799A的发明专利提供了一种抗冠状病毒的中药雾化剂，通过喷雾吸入药油，只可在鼻腔和气管内形成高浓度气态小环境而快速弑灭病毒，并不能在鼻黏膜表面形成药物层，阻断病毒入侵。公开号为CN112569331A的发明专利提供了一种预防新冠病毒的中药组合物及制备方法和应用，把中药组合物制备成香囊，但当中有效成分的挥发性有限，且在空气中容易扩散，难以维持较高的有效浓度。公开号为CN111280526A的发明专利提供了用于预防流感病毒和/或新型冠状病毒引起的肺炎的口罩及其制备方法，把中药组合物填充於口罩内的中药夹层包中，但口罩不能起到全方位、全时段的保护。
发明内容
本发明的第一目的在于提供一种口和/或鼻用药物组合物，以重量份计，其活性成分包括如下药物挥发油：
广藿香油15～30份、丁香油10～20份、草果油1～5份、生姜油10～20份以及益智仁油10～20份。
本发明的第二目的在于提供制备如上所述药物组合物的方法，包括：
a)分别制备各所述药物挥发油，并将它们混合在一起；以及
b)任选地制备所需剂型。
本发明的第三目的在于提供如上所提及的活性成分在制备用于防治SARS-CoV-2的药物中的应用。
本发明的有益效果为：
本发明所提供的药物组合物在对抗新冠病毒(即SARS-CoV-2)方面具有协同增效作用。其安全性高、药效好，主要活性成分为挥发油，尤其适用于作为口鼻保护剂使用，一方面利用挥发油自动挥发在鼻腔和气管内形成气态小环境而快速弑灭病毒，同时挥发油通过鼻腔粘膜的药物层杀灭沉积在鼻腔的病毒并抑制其对细胞的入侵，从而形成鼻腔的有效防线。尤其适用于封闭场所，达到全方位、全时段的保护。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为广藿香的指纹图谱[1：β-广藿香烯(3.03％)；2：石竹烯(1.43％)；3，6：α-愈创木烯(7.47％,2.26％)；4：β-蛇床烯(6.13％)；5：α-广藿香烯(5.22％)；7，10：α-布藜烯(9.93％,3.39％)；8：2,11-Dioxatetracyclo[4.3.1.1(3,10).0(6,9)]undec-4-ene,3,7,7,10-tetramethyl-(2.26％)；9：石竹素(1.90％)；11：广藿香醇(48.81％)]；
图2为草果的指纹图谱[1：α-蒎烯(2.30％)；2：β-蒎烯(2.51％)；3：α-水芹烯(7.56％)；4：p-伞花烃(1.61％)；5：桉叶油醇(44.04％)；6：(Z)-β-罗勒烯(1.73％)；7：4-Menthen-8-ol(2.50％)；8：(Z)-柠檬醛(3.66％)；9：香叶醇(1.81％)；10：8-methyl-1-decene(5.54％)；11：(E)-柠檬醛(5.42％)；12,13：4-propylbenzaldehyde(9.42％,1.52％)；14：橙花醇(2.14％)；15：橙花叔醇(1.67％)]；
图3为丁香的指纹图谱[1：丁香酚(75.59％)；2：石竹烯(3.73％)；3：异丁香酚(19.36％)]；
图4为姜黄的指纹图谱[1：桉叶油醇(1.80％)；2：benzene,1-(1,5-dimethyl-4-hexenyl)-4-methyl-(6.20％)；3：姜烯(10.50％)；4：异石竹烯(2.15％)；5：β-倍半水芹烯(9.49％)；6：芳姜黄酮(16.87％)；7：姜黄酮(31.62％)；8：姜黄新酮(12.65％)]；
图5为生姜的指纹图谱[1：α-蒎烯(2.39％)；2：莰烯(9.47％)；3：β-香叶烯(1.29％)；4：桉叶油醇(13.44％)；5：龙脑(2.69％)；6：石蜡烯(1.22％)；7：(Z)-柠檬醛(5.59％)；8：(E)-柠檬醛(5.40％)；9：benzene,1-(1,5-dimethyl-4-hexenyl)-4-methyl-(4.01％)；10：吉马烯D(1.52％)；11：姜烯(26.53％)；12：α-菌绿烯(4.87％)；13：β-紅沒藥烯(4.62％)；14：β-倍半水芹烯(8.76％)；15：α-蛇床烯(1.98％)]；
图6为苍术的指纹图谱[1：α-蒎烯(2.06％)；2：α-水芹烯(1.91％)；3：6S-2,3,8,8-tetramethyltricyclo[5.2.2.0(1,6)]undec-2-ene(1.75％)；4：六甲基苯(2.72％)；5：bicyclo[5.3.0]decane,2-methylene-5-(1-methylvinyl)-8-methyl-(1.44％)；6：石竹烯(1.48％)；7,8：γ-蛇床烯(2.29％,4.84％)；9：β-雪松烯(1.14％)；10：α-榄香烯(2.70％)；11：γ-榄香烯(6.60％)；12：α-古香油烯(1.50％)；13：茅苍术醇(6.22％)；14：β-桉叶醇(16.15％)；15：3,3'-diamino-4,4'-biphenyldiol(20.63％)；16：4-联苯甲醛(14.45％)]；
图7为檀香的指纹图谱[1：4-ethoxyphenyl acetamide(2.65％)；2：葛缕酮(4.11％)；3：7-propylidene-bicyclo[4.1.0]heptane(2.26％)；4：雪松醇(2.80％)；5：异长叶烷酮(4.23％)；6：α-雪松醇(8.70％)；7：蛇床二烯(2.07％)；8：cycloheptyl N.N-dipropylphosphoramidocyanidate(8.86％)；9：肉豆蔻酸异丙酯(11.51％)；10：2-methyl-3-phenylindole(2.88％)；11：methyl abiet-8-en-18-oate(16.62％)；12：脱氢松香酸甲酯(10.16％)]；
图8为益智仁的指纹图谱[1：百里香素(13.50％)；2：桉叶油醇(8.79％)；3：γ-松油烯(2.10％)；4：蛇床二烯(2.27％)；5：α-copaene-8-ol(2.37％)；6：β-花柏烯(3.60％)；7：雅槛蓝油烯(11.52％)；8：異戊酸異丙酯(7.27％)；9：γ-杜松烯(3.72％)；10：花柏酮(8.43％)]；
图9为莪术的指纹图谱[1：桉叶油醇(4.79％)；2：β-樟腦(1.27％)；3：β-石竹烯(3.82％)；4：莪术烯(3.50％)；5：β-花柏烯(1.72％)；6：3-Methyl-4,5,6,6a-tetrahydro-1H-pentalen-2-one(38.10％)；7：α-石竹烯(2.70％)；8：桉油烯醇(1.66％)；9：別香橙烯(2.01％)；10：(3E,7E)-10-Isopropenyl-3,7-cyclodecadien-1-one(14.37％)]；
图10为广藿香、草果、丁香、姜黄、生姜、苍术、檀香、益智仁和莪术的体外抗病毒活性结果。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有说明，用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导，随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本发明中所使用的术语“含有”、“包含”和“包括”是同义词，其是包容性或开放式的，不排除额外的、未被引述的成员、元素或方法步骤。
本发明中用端点表示的数值范围包括该范围内所包含的所有数值及分数，以及所引述的端点。
本发明中涉及浓度数值，其含义包括在一定范围内的波动。比如，可以在相应的精度范围内波动。比如2％，可以允许±0.1％范围内波动。对于数值较大或无需过于精细控制的数值，还允许其含义包括更大波动。比如100mM，可以允许±1％、±2％、±5％等范围内的波动。涉及分子量，允许其含义包括±10％的波动。
本发明中，涉及“多个”、“多种”等描述，如无特别限定，指在数量上指大于等于2。
本发明中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本发明中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本发明保护范围的限制。本发明中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可选”各自独立。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。除非和本申请的发明目的和/或技术方案相冲突，否则，本发明涉及的引用文献以全部内容、全部目的被引用。本发明中涉及引用文献时，相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本发明中涉及引用文献时，被引用的相关技术特征的举例、优选方式也可作为参考纳入本申请中，但以能够实施本发明为限。应当理解，当引用内容与本申请中的描述相冲突时，以本申请为准或者适应性地根据本申请的描述进行修正。
本发明涉及口和/或鼻用药物组合物，包括如下药物挥发油：广藿香油、丁香油、草果油、生姜油以及益智仁油。
优选的，以重量份计，其活性成分包括如下药物挥发油：
广藿香油15～30份、丁香油10～20份、草果油1～5份、生姜油10～20份以及益智仁油10～20份。
优选的，以重量份计，其活性成分包括如下药物挥发油：
广藿香油20～25份、丁香油13～17份、草果油2～4份、生姜油13～17份以及益智仁油13～17份。
优选的，以重量份计，其活性成分包括如下药物挥发油：
广藿香油21～24份、丁香油14～16份、草果油2～4份、生姜油14～16份以及益智仁油14～16份。
在一些实施方式中，所述药物组合物的活性成分还包括苍术油。
苍术油为10～20重量份；或13～17重量份。
在一些实施方式中，所述药物组合物的活性成分中含有或不含有如下成分或其提取物中的一种或多种：
麻黄、苦杏仁、生薏苡仁、茅苍术、青蒿草、虎杖、马鞭草、干芦根、葶苈子、化橘红、陈皮、茯苓、桔梗、厚朴、人参、黄芪、白术、防风、金银花、贯众、佩兰、陈皮、芦根、板蓝根、苏梗、大黄、姜黄、檀香以及莪术。
活性成分可以以有效发挥其抗病毒作用的量存在。在一些实施例中，按组合物的总重计，所述药物组合物包括约0.01重量％到约99.9重量％、约0.1重量％、约0.5重量％、约1重量％、约2重量％、约5重量％、约10重量％、约20重量％、约30重量％、约40重量％、约50重量％、约60重量％、约70重量％、约80重量％、约90重量％的活性成分。
“口和/或鼻用药物组合物”意指适于经口和/或鼻递送的组合物，包含口和/或鼻内递送。口和/或鼻用组合物的具体形式不受限制。在一些实施例中，口和/或鼻用组合物呈溶液、悬浮液、分散液、乳剂或凝胶的形式。在一些实施例中，组合物呈油性液体的形式。在一些实施例中，组合物是无水的或不含水的(如本文所使用的，“不含水”意味着，组合物在没有水的情况下配制，但可能存在痕量)。在一些实施例中，组合物包括不含水的半固态相。在一些实施例中，组合物是亲水凝胶或乳胶剂(即并入凝胶基质中的乳剂)。在一些实施例中，组合物是疏水凝胶，如油凝胶或有机凝胶。在一些实施例中，组合物包括具有小分子量有机胶凝剂(例如，<900Da)和/或聚合胶凝剂的三维粘弹性凝胶。在一些实施例中，组合物是可涂抹的。
在一种实施方式中，本发明的药物为简单混合物。
在一些实施方式中，所述的药物组合物为气雾剂、凝胶剂、膏剂、嗽口水、洗鼻剂和巴布剂中的任一种。
气雾剂系指药物与适宜抛射剂封装于具有特制阀门系统的耐压容器中制成的的制剂，使用时，借助抛射剂的压力将内容物以定量或非定量喷出，药物喷出多为雾状气溶胶，其雾滴一般小于50μm。
凝胶剂系指药物与能形成凝胶的辅料制成溶液、混悬或乳状液型的稠厚液体或半固体制剂。优选其为水性凝胶，其所用基质例如丙烯酸类、壳聚糖衍生物、海藻酸、改性纤维素、卡波姆。卡波姆进一步可选为卡波姆934、卡波姆940、卡波姆941等。凝胶剂中常含有pH调节剂，优选三乙醇胺、NaOH等。
膏剂优选为软膏、乳膏剂或贴膏剂。
巴布剂(cataplasma)是一种新型的外用制剂，系指将药物溶解或混合于水溶性高分子材料基质中，摊涂于裱背材料上，供皮肤贴敷的外用剂型。
所述的药物组合物还包含(i)～(v)中的至少一种：
(i)亲脂性或部分亲脂性媒剂；
(ii)表面活性剂；
(iii)卡波姆和/或三乙醇胺；
(iv)选自如下的药物活性组分：维生素、止痛剂、抗炎剂、止痒剂、退热剂和麻醉剂；和
(v)水(优选蒸馏水)。
在一些实施方式中，所述亲脂性或部分亲脂性媒剂选自：桉树油、蓖麻油、氢化蓖麻油、大豆油、芝麻油、花生油、甘油、石蜡、羊毛脂、脂肪酸酯、中链甘油三酯、脂肪酸甘油酯、聚乙二醇以及磷脂中的一种或多种。
表面活性剂选自阴离子、阳离子、两性和非离子表面活性剂，包含但不限于：杏仁油PEG-6-酯、卵磷脂、多元醇的脂肪酸酯、山梨聚糖的脂肪酸酯、聚氧乙烯山梨聚糖的脂肪酸酯、聚氧乙烯的脂肪酸酯、蔗糖的脂肪酸酯、聚甘油的脂肪酸酯、油酰基聚氧甘油酯、油酰基聚乙二醇甘油酯、山梨糖醇、甘油、聚乙二醇以及聚乙二醇甘油脂肪酸酯中的一种或多种。
如果存在，则表面活性剂可以以有效发挥表面活性剂性质的量存在。在一些实施例中，按组合物的总重计，组合物包括约1重量％到约20重量％、约1重量％到约10重量％、约1重量％到约5重量％、约4重量％或约2重量％的表面活性剂。在一些实施例中，按组合物的总重计，组合物包括1重量％到20重量％、1重量％到10重量％、1重量％到5重量％、4重量％或2重量％的表面活性剂。
各种维生素可包括在本发明的局部组合物中。可以使用例如维生素A和其衍生物、抗坏血栓、维生素B、生物素、泛酸、维生素D和其混合物。维生素E、醋酸生育粉和衍生物也可使用。
安全和有效量的抗炎剂可以加入本发明的组合物中，优选量是组合物量的约0.1％～约10％，更优选约0.5％～约5％。本发明组合物中使用的准确的抗炎剂量取决于使用的具体抗炎剂，因为这类药剂效力变化很大。
抗炎剂包括但不限于皮质甾类，例如可使用氢化可的松、羟曲安西龙、α-甲基地塞米松、地塞米松-磷酸酯、氯地米松二丙酸酯、氯倍他索戊酸酯、羟泼尼缩松、去氧米松、去氧皮脂酮乙酸酯、地塞米松、双氯松、双氟拉松二乙酸酯、双氟可龙戊酸酯、fluadrenolone、氯氟缩松、氟氢可的松、氟米松戊酸酯、fluosinolone acetonide、氟轻松醋酸、氟可丁丁基酯、氟可龙、氟泼尼定(fluprednylidene)醋酸酯、氟氢缩松、哈西编松、氢化可的松醋酸酯、氢可的松丁酸酯、甲泼尼松、曲安缩松、可的松、可托多松、flucetonide、氟氢可的松、difluorosone diacetate、fluradrenolone acetonide、甲羟松、安西法尔、安西非特、倍他米松和其剩余酯、氯泼尼松、氯泼尼松醋酸酯、氯可托松、Clescinolone、双氯松、双氟泼尼酯、氟氯缩松、氟泼尼松、氟米龙、氟培龙、氟泼尼松、氢可的松戊酸酯、氢可的松环戊基丙酸酯、氢可他酯、甲泼尼松、帕拉米松、泼尼龙、泼尼松、倍氯米松二丙酸酯、曲安西龙，和其混合物。
有用的麻醉或止痒药选自利多卡因、盐酸利多卡因、盐酸丁哌咔因、盐酸氯普鲁卡因、盐酸地步卡因、盐酸依替卡因、盐酸甲哌卡因、丁卡因、盐酸丁卡因、盐酸达克罗宁、盐酸己卡因、苯佐卡因、苄醇、苦味酸对氨苯酸丁酯、樟脑、樟脑酸化的间甲酚、地步卡因、盐酸地步卡因、盐酸异喹卡因、盐酸苯海拉明、杜松焦油、甲醇、苯酚、苯酚钠、盐酸丙吗卡因、间苯二酚和其混合物。
组合物中还可添加公知的有利于药效发挥/改善使用体验的组分，例如：
芳香剂：例如苯甲醛(如樱桃、杏仁)；柠檬醛；甲亢平；癸醛(桔子、柠檬)；C-8醛、C-9醛和C-12醛(柑桔果实)；甲苯甲醛(樱桃，杏仁)；2，6-二甲基辛醛(绿色果实)；2-十二烯醛(柑桔属，桔子)。这些芳香剂的混合物也可使用。
防腐剂、抗氧化剂、颜料。
保湿剂/增湿剂：本发明的组合物还可含有一种或多种保湿剂/增湿剂。可以使用各种保湿剂/增湿剂，使用量约1％～约10％，更优选约2％～约8％，最优选约3％～约5％。这些物质包括脲；胍；乙醇酸和乙醇酸盐(如铵和季烷基铵)；乳酸和乳酸盐(如铵和季烷基铵)；多羟基醇，如山梨醇、甘油、己三醇、丙二醇、己二醇，等等；聚乙二醇；糖和淀粉；糖和淀粉衍生物(如烷氧基化的葡糖)；D-泛酰醇；透明质酸；乳酰胺单乙醇胺；乙酰胺单乙醇胺；和其混合物。
本发明还提供制备如上所述药物组合物的方法，包括：
a)分别制备各所述药物挥发油，并将它们混合在一起；以及
b)任选地制备所需剂型。
在一些实施方式中，制备各药物挥发油的方法选自蒸馏法、溶剂提取法、超声波辅助萃取法、微波萃取法或超临界流体萃取法。
本发明还涉及如上所述的药物组合物(或活性成分)在制备用于防治SARS-CoV-2的药物中的应用。
可以以任何适合的量施用所述药物组合物。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用约0.1ml到约1ml，如约0.1ml到约0.3ml、约0.15ml到约0.25ml或约0.175ml到约0.225ml。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用0.1ml到1ml，如0.1ml到0.3ml、0.15ml到0.25ml或0.175ml到0.225ml。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用约0.1ml、约0.15ml、约0.2ml、约0.25ml或约0.3ml。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用0.1ml、0.15ml、0.2ml、0.25ml或0.3ml。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用约0.2ml或更少。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用0.2ml或更少。
在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用的组合物的量包括约0.001g到约0.3g试剂，如约0.005g到约0.2g试剂或约0.01g到约0.1g试剂。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用的组合物的量包括0.001g到0.3g试剂，如0.005g到0.2g试剂或0.01g到0.1g试剂。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用的组合物的量包括约0.001g、约0.004g、约0.005g、约0.01g、约0.02g、约0.03g、约0.04g、约0.05g、约0.09g或约0.1g试剂。在一些实施例中，向口内、一个或两个鼻孔每鼻孔施用的组合物的量包括0.001g、0.004g、0.005g、0.01g、0.02g、0.03g、0.04g、0.05g、0.09g或0.1g试剂。
在一些实施例中，向口内、一个或两个鼻孔每鼻孔每天一次施用组合物。在一些实施例中，向口内、一个或两个鼻孔每鼻孔每天两次施用组合物。在一些实施例中，向口内、一个或两个鼻孔每鼻孔每天三次、四次、五次或六次施用组合物。
下面将结合实施例对本发明的实施方案进行详细描述。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，优先参考本发明中给出的指引，还可以按照本领域的实验手册或常规条件，还可以参考本领域已知的其它实验方法，或者按照制造厂商所建议的条件。
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。
实施例
1.中药挥发油的制备：
参照《中国药典》(2015版，四部)通则2204项规定方法(甲法)操作。称取各药材粗粉(或碎片)适量，置于1000mL圆底烧瓶中，加入适量(8倍料液比)蒸馏水，连接挥发油提取器与回流冷凝管，自冷凝管上端加水使充满挥发油测定器的刻度部分，并溢流入烧瓶时，置电热套上缓缓加热至沸，并保持微沸约6小时，至测定器中油量不再增加，停止加热，收集蒸馏液，并静置冷却片刻后转移至离心管中，离心10分钟(10000转/分钟)，取挥发油层，读取油量并获得各样品的挥发油。结果广藿香、草果、丁香、姜黄、生姜、苍术、檀香、益智仁和莪术的得油率分别为0.42％、1.80％、10.67％、1.50％、0.06％、0.62％、1.30％、0.29％和0.21％。
2.中药挥发油指纹图谱的建立：
采用Agilent:GC-MS 7890A-5975C进行检测，①毛细管色谱柱：DB-1MS,30m,0.25mm i.d.,0.25μm film thickness；②进样口温度：250℃；③接口温度：280℃；④载气流速：1.0mL/min；⑤分流比：100:1；⑥升温程序：起始100℃，以每分钟8℃的速率升温至300℃，并保持5分钟；⑦质谱离子源温度：230℃；⑧四极杆温度：150℃。数据处理由GC-MS分析软件系统完成，通过检索NIST08质谱库，把各色谱峰与标准图谱进行匹配，并按峰面积归一化计算各色谱峰的相对百分含量。结果获得各中药挥发油的指纹图谱见图1-9。
3.抗新型冠状病毒细胞实验：
本实验委托广州呼吸健康研究院进行，所有实验操作均在BSL-3实验室内完成。
材料：受试药物(上述挥发油)；SARS-CoV-2临床分离株(GeneBank登录号为MT123290.1)，滴度为10-7TCID50/mL，使用病毒滴度为100TCID50/孔；Vero E6非洲绿猴肾细胞株，购自美国ATCC生物标准品资源中心，培养在含2％胎牛血清的RPMI 1640培养基中。
细胞毒性测试：于96孔细胞培养板上接种2×105个细胞/孔，培养16-20h至细胞长成单层，弃去培养基。将受试药物用完全培养基作连续4倍稀释，再按每孔100μL加入细胞中，即为加药组。同时设细胞对照组和阳性对照组，每组设4个复孔。细胞培养72h后，利用MTT法测定细胞存活量，从而計算受试药物对细胞的半数有毒浓度(CC50)。
体外抗病毒活性测定：于96孔细胞培养板上接种5×104个细胞/孔，培养16-20h至细胞长成单层，弃去培养基。将受试药物用完全培养基作连续4倍稀释，依次吸取50μL至96孔板中，各加病毒液50μL；同时设细胞对照组、模型组和阳性对照组，每组设4个复孔。细胞培养24h后，在倒置显微镜下观察并记录细胞形态变化(CPE)。细胞无病变为"–"，病变0-25％为"+"，病变25％-50％为"++"，病变50％-75％为"+++"，病变75％-100％为"++++"。根据观察结果用Reed-Muench法计算药物的半数抑制浓度(IC50)。最後，抗病毒活性以选择指数(SI)表示，SI的计算方法为CC50与IC50的比值，即SI＝CC50/IC50。结果表明，9种中药挥发油均具有抗新冠病毒活性(SI>1)，广藿香、草果、丁香、姜黄、生姜、苍术、檀香、益智仁和莪术的IC50(SI值)分为31.88μg/mL(1.96)、9.33μg/mL(1.95)、77.35μg/mL(1.95)、43.57μg/mL(3.22)、29.10μg/mL(1.99)、18.82μg/mL(1.86)、20.30μg/mL(1.94)、17.10μg/mL(1.95)和15.10μg/mL(1.16)(见图10)。
4.复方中药挥发油的制备及其抗新冠病毒活性
实施例1：按体积份数计，包括广藿香油23份、丁香油15份。
实施例2：按体积份数计，包括草果油3份、丁香油15份。
实施例3：按体积份数计，包括广藿香油23份、丁香油15份、生姜油15份。
实施例4：按体积份数计，包括广藿香油23份、丁香油15份、益智仁油15份。
实施例5：按体积份数计，包括广藿香油23份、益智仁油15份。
实施例6：按体积份数计，包括广藿香油23份、生姜油15份、益智仁油15份。
实施例7：按体积份数计，包括广藿香油23份、草果油3份、生姜油15份、益智仁油15份。
实施例8：按体积份数计，包括广藿香油23份、丁香油15份、生姜油15份、益智仁油15份。
实施例9：按体积份数计，包括广藿香油23份、丁香油15份、草果油3份、生姜油15份、益智仁油15份。
实施例10：按体积份数计，包括广藿香油23份、丁香油15份、草果油3份、生姜油15份、益智仁油15份、苍术油15份。
表1.实施例1-10的抗新冠病毒活性(IC50)
结果表明，实施例9和10的IC50低于活性最高的单方挥发油(草果)，表示该两种复方中药挥发油发挥协同增效作用，具有更佳的抗新冠病毒活性。
5.凝胶剂的制备
单方或复方中药挥发油：1-50重量份、卡波姆-940：8-40重量份、吐温-80：8-20重量份、甘油：160-400重量份、三乙醇胺：32-56重量份、纯净水：500-800重量份。取卡波姆-940置于40倍重量份纯净水中溶胀12h得半固体凝胶(Ⅰ)，取吐温-80和甘油加入到(Ⅰ)中，边加入边搅拌得稠膏(Ⅱ)，另加入三乙醇胺至稠膏(Ⅱ)中，并取单方或复方中药挥发油加入均匀搅拌得稠膏(Ⅲ)，最后向稠膏(Ⅲ)中缓慢加入纯净水，搅拌均匀。
6.软膏剂的制备
单方或复方中药挥发油：1-50重量份、液体石蜡：250-350重量份、羊毛脂：650-750重量份。液体石蜡和羊毛脂置于60℃水浴中加热搅拌至液体均匀透明，再加入单方或复方中药挥发油，搅拌均匀。
7.嗽口水的制备
单方或复方中药挥发油：1-50重量份、甘油：5-100重量份、聚山梨酯：0.5-50重量份、纯净水800-950重量份。将单方或复方中药挥发油和聚山梨酯于搅拌的条件下加入甘油中，充分搅拌后加入纯净水。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准，说明书及附图可以用于解释权利要求的内容。
Claims (12)
1.口和/或鼻用药物组合物，以重量份计，其活性成分包括如下药物挥发油：
广藿香油15～30份、丁香油10～20份、草果油1～5份、生姜油10～20份以及益智仁油10～20份；
所述药物组合物用于抗SARS-CoV-2病毒。
2.根据权利要求1所述的药物组合物，以重量份计，其活性成分包括如下药物挥发油：
广藿香油20～25份、丁香油13～17份、草果油2～4份、生姜油13～17份以及益智仁油13～17份。
3.根据权利要求1或2所述的药物组合物，其活性成分还包括苍术油10～20份。
4.根据权利要求3所述的药物组合物，所述苍术油为13～17份。
5.根据权利要求1、2、4任一项所述的药物组合物，其为气雾剂、凝胶剂、膏剂、洗鼻剂和巴布剂中的任一种。
6.根据权利要求1、2、4任一项所述的药物组合物，其还包含(i)～(iv)中的至少一种：
(i)亲脂性或部分亲脂性媒剂；
(ii)表面活性剂；
(iii)卡波姆和/或三乙醇胺；和
(iv)水。
7.根据权利要求6所述的药物组合物，所述亲脂性或部分亲脂性媒剂选自：桉树油、蓖麻油、氢化蓖麻油、大豆油、芝麻油、花生油、甘油、石蜡、羊毛脂、脂肪酸酯、中链甘油三酯、脂肪酸甘油酯、聚乙二醇以及磷脂中的一种或多种。
8.根据权利要求6所述的药物组合物，所述表面活性剂选自：杏仁油PEG-6-酯、卵磷脂、山梨聚糖的脂肪酸酯、聚氧乙烯山梨聚糖的脂肪酸酯、蔗糖的脂肪酸酯、聚甘油的脂肪酸酯、油酰基聚氧甘油酯、油酰基聚乙二醇甘油酯、山梨糖醇、甘油、聚乙二醇以及聚乙二醇甘油脂肪酸酯中的一种或多种。
9.制备权利要求1～8任一项所述药物组合物的方法，包括：
分别制备各所述药物挥发油，并将它们混合在一起。
10.根据权利要求9所述的方法，其还包括：制备所需剂型。
11.根据权利要求9或10所述的方法，制备各药物挥发油的方法选自蒸馏法、溶剂提取法、超声波辅助萃取法、微波萃取法或超临界流体萃取法。
12.权利要求1～4任一项中所述的药物组合物在制备用于抗SARS-CoV-2病毒的药物中的应用。
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